Tau is the major protein component of the characteristic intracellular protein aggregate of Alzheimer's disease and related disorders, the neurofibrillary tangle. Tau deposited in neurofibrillary tangles is markedly hyperphosphorylated, but the role of phosphorylation in influencing tau neurotoxicity has been difficult to dissect in conventional experimental systems. In preliminary experiments, we provide two lines of evidence supporting a critical role for phosphorylation in controlling tau toxicity in a transgenic Drosophila model of tauopathy. First, kinases and phosphatases comprise the major category of genetic modifiers identified in a forward genetic screen for modifiers of tau toxicty. These kinase and phosphatase modifiers include several enzymes known to phosphorylate or dephosphorylate tau in vitro, as well as novel kinases. Selective candidate testing also reveals that overexpression of the known tau kinases MARK, protein kinase A, and CDK5 enhances tau toxicity. Second, mutation of 14 proline-directed serine or threonine phosphorylation sites on tau, including the AT8, AT100, TG3, and PHF-1 sites, markedly attenuates tau toxicity. We now propose to investigate the role of proline-directed phosphorylation in determining tau neurotoxicity in detail. In Specific Aim 1 we will define the phosphorylation sites required for tau neurotoxicity by mutating sites individually or in small groups. These experiments will reveal if any individual phosphorylation site exerts a significant influence on tau toxicity, or if sites work primarily in concert. When the phosphorylation site(s) controlling tau toxicity have been defined, we will confirm the role of phosphorylation in determining toxicity by altering these site(s) to negatively charged amino acids to mimic phosphorylation. In Specific Aim 2 we will determine if the kinase modifiers we have identified through our genetic screens and candidate testing act on specific phosphorylation sites by determining if kinase modifiers are able to alter the toxicity of phosphorylation site mutant tau. Finally, in Specific Aim 3 we will investigate the role of our novel kinase modifiers in human neurodegenerative diseases characterized by deposition of abnormally phosphorylated and aggregated tau, including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy.